Successful adaptation of a crop
species is dependent upon the programming of critical growth stages so that the
plant can capitalize on favorable weather periods during the growing season.
Plants have evolved a variety of adaptive mechanisms that allow them to
optimize growth and development while coping with environmental stresses. These
mechanisms include seed and bud dormancy, photoperiod sensitivity, and
low-temperature response. Seed dormancy delays germination until after the
embryo has gone through an after-ripening period. The over-winter survival of
buds of many temperate zone trees and shrubs is dependent on a dormancy stage
that starts in the late summer or early fall and ends after exposure to an
extended period of cold or increasing day length in the spring. In addition to
trees, many other dicots and grasses have a photoperiod response that can
advance or delay flowering. Vernalization is a requirement for growth at low
temperatures before a plant will flower. Most winter annual and biennial plants
have a vernalization requirement. Low-temperature acclimation is an ability of
plants to cold acclimate when exposed to gradually decreasing temperatures
below a specific threshold. This is the most common mechanism that plants have
evolved for adapting to low-temperature stress and examples of plants with the
capacity to cold harden can be found in most species.

Types of Low Temperature Injury

There
are two types of injuries a plant can sustain through exposure to low
temperatures (Stushnoff et al. 1984).The first is chilling injury that occurs from approximately 20 to 0oC.The resultant injuries may include a variety
of physiological disruptions in germination, flower and fruit development,
yield, and storage life. Minor chilling stress at non-lethal temperatures is
normally reversible. Exposure to gradually decreasing temperatures above the
critical range can also result in hardening of plants that may reduce or
eliminate injury during subsequent exposure to low temperatures.

The
second type of injury is called freezing injury. This type of injury occurs
when the external temperature drops below the freezing point of water.Some varieties of plants that are susceptible
to chilling injury can be killed by the first touch of frost. At the other extreme,
many plants that are native to cold climates can survive extremely low
temperatures without injury (Levitt 1980). Plants may experience intracellular
freezing and/or extracellular freezing.Intracellular freezing damages the protoplasmic structure and the ice
crystals kill the cell once they grow large enough to be detected
microscopically. In extracellular freezing, the protoplasm of the plant becomes
dehydrated because a water-vapor deficit is created as cellular water is
transferred to ice crystals forming in the intercellular spaces. In some cases,
water can remain liquid as low as -47oC without nucleating and
forming ice. When nucleation of this supercooled water does occur,
intracellular ice forms suddenly resulting in death of the plant.

Types of Plants

Plants
can be grouped into three different classes according to their low-temperature
tolerance (Stushnoff et al. 1984). The first group includes frost tender plants
that are sensitive to chilling injury and can be killed by short periods of
exposure to temperatures just below freezing. They cannot tolerate ice in their
tissues and readily exhibit frost injury symptoms that include a water soaked
flaccid appearance with loss of turger followed by rapid drying upon exposure
to warm temperatures. Beans, corn, rice, and tomatoes are examples of plants in
this category.

Low-temperature
acclimation of plants in the second group allows them to tolerate the presence
of extracellular ice in their tissues. Their frost resistance ranges from the
broad-leafed summer annuals, which are killed at temperatures slightly below
freezing, to perennial grasses that can survive exposure to -40oC.
As temperatures decrease the outward migration of intracellular water to the
growing extracellular ice crystal causes dehydration stress that will
eventually result in irreversible damage to the plasma membrane, which is the
primary site of low-temperature injury. If ice nucleation does not occur at -3
to -5oC, supercooling may result in intracellular freezing and death
of individual cells.

The
final group is made up of very cold hardy plants that are predominantly
temperate woody species. Like the plants in the previous group, their lower
limits of cold tolerance are dependent on the stage of acclimation, the rate
and degree of temperature decline, and the genetic capability of tissues to
accommodate extracellular freezing and the accompanying dehydration stress.
Deep supercooling allows certain tissues in plants from this group to survive
low temperatures without the formation of extracellular ice. However, the most
cold hardy species do not rely on supercooling and can withstand temperatures
of -196 oC.

2. Plant Chilling Stress and Its Repercussions

Introduction

Most
crops of tropical origin as well as many of subtropical origin are sensitive to
chilling temperatures. This limits production areas and causes potential damage
during storage if they are exposed to low temperatures. The temperature below
which chill injury can occur varies with species and regions of origin, ranging
from 0 to 4oC for temperate fruits, 8oC for subtropical
fruits, and about 12oC for tropical fruits such as banana (Lyons
1973). Amongst the highest volume world food crops, maize (Zea mays) and rice (Oryza
sativa) are sensitive to chilling temperatures. Their growth and
development can be adversely effected by temperatures below 10oC
resulting in yield loss or crop failure. Christiansen and St. John (1981)
estimated annual losses of $60 million to the cotton industry due to chilling
temperature immediately following field planting. Chilling during the seedling
stage in cotton can reduce plant height, delay flowering and adversely affect
yield and lint quality. Seedlings can also suffer water stress and leaf
desiccation at chilling temperatures, floral initiation is inhibited at 7oC
and seed set is inhibited at 15oC. Other crops suffering stand loss,
delayed maturity, and reduced yield as a result of chilling after planting
include soybean (Glycine max L.),
lima bean (Phaseoluslunatus L.), cucurbits (Cucurbita sp.), tomato (Lycopersicon esculentum Mill.) pepper Capsicum annuum L.), eggplant (Solanum melongena L.), okra (Abelmoschus esculentus L.), and various
cereal crops.

Physiological
age, seedling development, and pre-harvest climate can also influence chilling
sensitivity. Freshly imbibed seeds of chill-sensitive species tend to be very
sensitive, as does the pollen development stage. Fruits maturing at high
temperature are more susceptible than those maturing at lower temperatures.
Post-harvest storage at lower temperatures is commonly used to extend the
storage life of fruits and vegetables. Tropical and subtropical plants however
are often subject to physiological damage and loss of quality due to chill
injury under these storage conditions. The severity of injury to
chill-sensitive tissues tends to increase with decreasing temperatures and with
length of low-temperature exposure.

Chilling
has been found to change the entire metabolic system of the cell with some
processes recovering quickly and others only slowly. Chilling affects the
entire internal environment of each cell and each molecule within the cells.
Enzymatic reactions, substrate diffusion rates, and membrane transport
properties are all affected. Chilling injury is therefore likely a direct
consequence of these effects (Kratsch and Wise 2000).

Amelioration
of chilling injury

Avoidance:
To avoid chilling injury, planting dates can be altered though this is often
difficult because of its effect on later development of the plant. To overcome
this problem, cultivars have been bred for early vigor and maturity. In the
case of stored fruits and vegetables, maintenance of appropriate storage
temperatures is essential to avoid chilling injury. Investigations have also
been undertaken to examine synthetic plant growth regulators for the protection
of chilling sensitive crops (Li 1989).

Temperature conditioning:
Low-temperature ‘hardening’ allowing tolerance to chilling temperatures appears
to have little effect although some sensitivity to ‘slight chilling’ can be
reduced by exposure to temperatures slightly above the chilling range. It also
appears that chilling injury to stored fruits and vegetables can be ameliorated
by warm temperatures if they are imposed before tissue degeneration becomes
advanced. Other treatments such as waxing, fungicides, hormones, and
antioxidants have produced variable results that have been dependent upon the
species and treatment conditions (Lyons 1973).

Duration:Ultrastructural-chilling injury
increases with time and with prolonged exposure the injury becomes
irreversible. It is therefore important to minimize the time of chilling
temperature exposure.

Relative humidity:
High (100%) relative humidity has been found to protect chloroplasts from chill
injury, an effect that is enhanced by darkness.

Theories of chilling injury

Early
research focused on chilling causing an imbalance in plant physiological
processes. Chilling was found to affect O2 evolution, organic acids,
sugars, polyphenols, phospholipids, protein, and ATP. Research indicates that
chilling stress in sensitive plants changes most chemical entities. There is
evidence of accumulation of toxins such as ethanol and acetaldehyde. Although
many altered processes involve key metabolites; it is difficult to separate the
critical chilling-sensitive metabolic processes from those that are byproducts
of metabolic disruptions or of ultrastructural breakdown. Ion leakage due to
membrane permeability changes has often been reported in chill sensitive
plants. Phase transition of the lipid portion of the cellular membranes has
also received considerable attention as the primary
response to chilling temperatures (Lyons 1973).

Ultrastructural changes: On
an ultrastructural level, several changes have been associated with chilling
injury. Although there are a number of variables affecting chill injury, the
ultrastructural symptoms are very similar across species. Ultrastructural
symptoms of chilling injury become evident before obvious physical symptoms are
visible. These include changes to chloroplasts, mitochondria and membranes
associated with these organelles and the vacuoles (Christiansen and St. John
1981). The symptoms include swelling and disorganization of the chloroplasts
and mitochondria, reduced size and number of starch granules, dilation of
thylakoids and unstacking of grana, formation of small vesicles of chloroplast
peripheral reticulum, lipid droplet accumulation in chloroplasts, and
condensation of chromatin in the nucleus (Kratsch and Wise 2000).

Chloroplasts
are the first and most severely affected organelle. Irradiance during chilling
greatly exacerbates the resulting injury. Chilled plants in darkness have been
found to remain green and, except for starch depletion, chloroplasts appear
normal. In the presence of light, however, chlorophyll becomes bleached, lipid
droplets accumulate, and thylakoids degenerate. Mitochondria appear more
resistant to chilling temperature but an immediate effect of low temperature on
chilling-sensitive species is a suppression of mitochondrial activity. Electron
micrographs of chilled sweet potato roots revealed that the mitochondria had a
swollen appearance due to the release of phospholipids from the inner and outer
membranes during storage at chilling temperatures. The capacity to bind
phospholipids was also greatly decreased.

Membrane permeability and
phase transition: Measures of solute leakage or ion
permeability have provided evidence of increased membrane permeability in
response to chilling. The plasma membrane is often considered the primary site
of freezing injury and electrolyte leakage. Early work indicated that plants
originating in warm climates tend to have more saturated fatty acids in their
membrane lipids. More recent work on mitochondrial membranes has shown that
membranes do undergo a physical phase transition from a flexible
liquid-crystalline to a solid-gel structure at 10 to 12oC, which
coincides with the temperature sensitivity range of species of tropical origin.
Fruits of several apple cultivars have been observed to undergo phase
transition in the 3 to 10oC range suggesting the same mechanism of
chilling injury as found in tropical species. The correlation between fatty
acid composition and temperature induced phase transition is, however, not
precise. It may be that other membrane components such as sterols also play a
role.

It
is possible that the phase transition of cellular membranes could account for
the entire range of physiological and metabolic changes associated with
chilling injury. Increased membrane permeability could lead to an altered ion
balance and also to the ion leakage observed from chilling of sensitive
tissues. Phase transition could result in conformational changes in membrane bound
enzymes and account for the observed discontinuities in the function of many
enzyme systems. This may cause an imbalance between membrane bound and
non-membrane bound systems. Over time the cells inability to cope with
increased concentrations of metabolites could result in injury. Different
tolerances to these metabolites could explain why some cultivars are more
resistant to damage while still undergoing phase transition. Imbalances in
metabolism, accumulation of toxic compounds, and increased permeability could
all be the result of temperature-induced phase transition (Lyons 1973).

The
contribution of unsaturated fatty acids in cell membrane lipids has been
discussed for many years in relation to chilling sensitivity. Nishida and
Murata (1996) have shown that chilling injury can be manipulated by modulating
levels of unsaturation of fatty acids by the action of acyl-lipid desaturases
and glycerol-3-phosphate acyltransferase. Lyons (1973) proposed that
temperature induced phase transition of membrane lipids may play a primary role
in chilling sensitivity of plants. Continued exposure to chilling temperatures
would result in phase separated membranes becoming incapable of maintaining
ionic gradients resulting in metabolic disruption and eventual cell death. A
positive correlation has been found between chilling sensitivity of herbaceous
plants and the level of saturated and trans-monounsaturated
molecular species of phosphatidylglycerol(also termed high-melting-point molecular species) in thylakoid membranes.
However, there is still a question of how directly these high-melting-point
molecular species relate to chilling sensitivity in plants. Growth at low
temperature generally increases the degree of unsaturation of membrane lipids,
which compensates for the decrease in fluidity caused by the lower temperature.
This increased unsaturation is also correlated with the sustained activity of
membrane-bound enzymes at low temperature. The unsaturation of membrane lipids
is therefore considered critical for the functioning of biological membranes
and the survival of plant cells at low temperature. However, since low
temperature is also known to induce or alter the expression level of a large
number of genes it is not clear if the association between membrane lipid
unsaturation and chilling tolerance is a cause or effect relationship.

Recently the role of unsaturation of membrane
lipids in chilling tolerance and in response to low temperature has been
reexamined using mutant and transgenic lines (Nishida and Murata 1996). In this
way unsaturated fatty acids can be manipulated independent of temperature so
that their individual effects can be evaluated. Tobacco was transformed with
squash and Arabidopsis phosphatidylglycerol
(PG) species found in thylakoid membranes. Squash has low levels of cis-unsaturated PG while Arabidopsis has relatively high levels
of cis-unsaturated PG. It was found
that tobacco transformed with squash PG was more chilling sensitive and tobacco
transformed with Arabidopsis PG was
the most chilling resistant, as measured by photosynthesis at 1oC
under strong illumination. These results indicate that chilling sensitivity can
be manipulated by altering the level of unsaturated PG in the chloroplasts.
These and other experiments have shown that unsaturation of membrane lipids
protect the photosystem II complex from low-temperature photoinhibition by
accelerating recovery from the photoinhibited state. However, it is likely that
other factors such as accumulation of polyols and amino acids, or their
derivatives, contribute to chilling sensitivity in plants. Some specific
proteins may also be responsible for chilling tolerance.

Alteration of
intracellular pH:
Yoshida
et al. (1999) noted that intracellular pH was, in part, actively controlled by
H+-transport from the cytoplasm to the vacuole catalyzed by H+-ATPase
located on the vacuolar membrane in mung bean (Vigna radiata L.),
which is a very chilling-sensitive species. The vacuolar H+-ATPase
is extremely sensitive to low temperature and is preferentially inactivated
upon exposure to chilling temperatures. This inactivation occurs much earlier
than the symptoms of cell injury and the decrease in enzyme activity associated
with plasma membranes, endoplasmic reticulum, and mitochondria. Cold-induced
inactivation of H+-ATPase also occurs in chilling sensitive rice.
Cold-induced suppression of proton transport disrupts cytoplasmic homeostasis
and causes a change in the pH. The chilling sensitivity of cultured mung bean
cells changed markedly during the growth cycle and a close relationship was
found between sensitivity of the cells and of H+-ATPase to the cold.
Cold-induced inactivation of the vacuolar H+-ATPase was closely
linked to acidification of the cytoplasm and the corresponding alkalization of
the vacuoles suggesting a passive release of H+ ions across the
vacuolar membrane. The susceptibility of vacuolar H+-ATPase to low
temperature in vivo was found to be markedly different between
chilling-sensitive and chilling-resistant species. In contrast to the H+-ATPases
of chilling-sensitive species like mung bean and kidney bean (Phaseolus
vulgaris),
the H+-ATPases of the chilling-tolerant species such as pea (Pisum sativum)
and broad bean (Vicea faba) were very stable over long periods of
low-temperature exposure. The molecular structures of the 16 kDa proteolipids
from the two types of H+-ATPase appeared to be very different.
Low-temperature-induced pH reduction of the cytoplasm caused by inactivation of
vacuolar H+-ATPase may therefore be the cause of extreme
chilling-sensitivity.

Conclusion

Plants are uniquely adapted to their native environment
through developmental programming and the particular composition and
conformation of their molecular components is optimized within each species for
maximum competitive ability. These differences in adaptation result in the wide
range of cellular disturbances that have been observed when these plants are
moved to cooler environments. Changes in enzyme reactions, substrate diffusion
rates, membrane properties, and cytoplasmic pH affect the entire metabolic
system of cells subjected to chilling stress. The resulting injury depends on
the duration of exposure and on the individual species, or variant, being
observed.

3. The Physiological and Agronomic Repercussions of
Freezing Stress

Low-temperature
response mechanisms

Figure 1. Changes in cold
hardiness of Norstar winter wheat for the period September to May. The
primary factors responsible for these changes are shown at the bottom of the
graph.

Plants
have adapted two mechanisms to protect themselves from damage due to below
freezing temperatures.Supercooling is a
low-temperature tolerance mechanism that is usually associated with acclimated
xylem parenchyma cells of moderately hardy woody plants. When sources of ice
nucleation are absent, pure water can supercool or remain unfrozen to its
homogeneous nucleation point of approximately -40oC. The initiation
of freezing at the limit of supercooling occurs suddenly and is accompanied by
an exotherm that can be detected by thermal analyses of plant tissues. Plant
tissues suffer irreversible damage once ice nucleation of supercooled water
occurs and the distribution in nature of tree species with the ability to deep
supercool is normally restricted to regions where winter temperatures are
warmer than -40oC (George et al. 1982).

The
second and most common low-temperature response mechanism is acclimation.
Low-temperature acclimation is a gradual process during which there are changes
in just about every measurable morphological, physiological, and biochemical
characteristic of the plant.These
changes are determined by genotype x environment interactions that are quite
complex and not clearly understood. They have been studied most extensively in cereals where a wide range in
genetic potential and the availability of unique cytogenetic stocks has allowed
for novel approaches to investigations at the molecular and whole plant level.
Potential gene donors have been evaluated for use in interspecific transfers
and the control of alien (donor species) low-temperature gene expression has
been studied in a variety of backgrounds. A survey of the published research in
these areas has allowed us to construct a field validated winter
survival model that successfully simulates the over winter changes in
low-temperature tolerance of a wide range of genotypes (Fowler et al. 1999). Consequently, this review will focus
mainly on the genetic systems that winter cereals have evolved for
low-temperature adaptation, the regulation of these systems, and their complex
interaction with the environment.

Low-Temperature Acclimation in Winter
Cereals

Figure 3. Relationship
between average daily crown temperature (0 to 8oC) and low-temperature tolerance
of Norstar winter wheat during acclimation at a constant temperature before
vernalization saturation (LT50 for Norstar = - 24oC when fully acclimated).

Figure 5. The
effect of exposure to a constant temperature on low-temperature tolerance
of Norstar winter wheat (Norstar LT50 = - 24oC when fully
acclimated at day 0).

When growth starts in the early
fall, winter cereal plants will not survive subfreezing temperatures much
better than spring cereal plants.However, winter cereals grown under cool fall temperatures will cold acclimate
or 'harden off'.For example, in Saskatchewan
(western Canada), the minimum survival temperature for 'Norstar' winter wheat
is normally near -3oC at the beginning of September and -19oC
or lower by the end of October (Figure 1).

Under field conditions in western Canada, eight to 12 weeks of fall
growth is usually required for the full development of cold hardiness in winter
cereals.The first four to five weeks is
a period of active growth that takes place when average daily soil

temperatures at crown depth are above 9oC. Both the cold
acclimation process and winter survival require energy and this period of warm
temperature allows for the establishment of healthy vigorous plants (Figure
2).Plants with well-developed crowns
before freeze-up are in the best position to withstand the rigors of winter and
regenerate roots and leaves in the spring. However, plants that enter the
winter with two to three leaves are usually not seriously disadvantaged.

Cold acclimation of winter wheat plants begins once fall temperatures
drop below approximately 9oC. A translocatable substance that
promotes cold acclimation is not produced when winter wheat plants are exposed
to acclimating temperatures (Limin and Fowler 1985).Consequently, the cold-hardiness level of
different plant parts, such as leaves, crowns and roots, is dependent upon the
temperature to which each part has been exposed. Because the crown contains
tissues that are necessary for plant survival, it is the soil temperature at
crown depth that determines critical cold-acclimation rates.

Plant growth slows
considerably at temperatures that promote cold acclimation. In the field, soil
temperatures gradually decrease as winter approaches and four to seven weeks at
temperatures below 9oC is usually required to fully cold-harden
plants. Cold acclimation during this period is dependent upon crown
temperatures and the rate of acclimation increases dramatically as temperature
drop from 9 to 0oC (Figure 3). Exposure of winter wheat crowns to soil
temperatures above 9oC during this period results in a rapid loss of
cold hardiness. The rate of dehardening is dependent upon the temperature to
which the crown is exposed (Figure 4).At this stage, plants that have been exposed to crown temperatures above
9oC will resume cold acclimation once they return to temperatures
below 9oC.

Winter wheat normally does not realize its maximum cold hardiness
potential until after the soil is frozen in the late fall.In Saskatchewan, full acclimation is usually
achieved by the middle to the end of November (Figure 1). Once cold acclimation
has been completed, winter wheat can maintain a high level of cold hardiness
provided crown temperatures remain below freezing. In the fall, winter wheat
will cold acclimate when exposed to crown temperatures colder than 9oC.
However, prolonged exposure of acclimated plants to winter temperatures above
freezing results in the transition of the plant from the vegetative to the reproductive
phase and a gradual loss of cold hardiness (see the next section for more
details on these changes). The warmer the crown temperature during the winter,
the shorter the period that maximum levels of cold hardiness can be maintained
and, once started, the more rapid the rate of decline in cold hardiness.

Death of the crown
tissue will result if the soil temperature falls below the plants minimum
survival temperature (Figure 1).Exposure of winter cereal plants to crown temperatures that are 2 to 3oC
warmer than their minimum survival temperature will cause immediate damage and
a reduction in cold hardiness (Fowler et al. 1999). Longer periods of exposure
to temperatures approaching the minimum survival temperature can quickly reduce
the plant's ability to tolerate cold stress. The expected LT50 for different exposure
times (T) to constant temperature can be calculated from Equation 1 (Fowler et
al. 1999).

LT50(T) = LT50(0) + 5.72 + 1.53 *
ln(T)[Eq. 1]

Where T is the
number of days that plants are exposed to a constant low-temperature
stress.LT50(0) is determined
using a series of test temperatures where the low-temperature stress is removed
as soon as the crown samples are exposed to a predetermined minimum temperature
(day 0 in Figure 5). For example, fully acclimated Norstar winter wheat will normally survive
to -24.0oC in a controlled-freeze test where plant samples are
gradually cooled at a rate of 2 to 6oC hr-1 and removed
as soon as they reach a predetermined temperature (day 0 in Figure 5). However, two days exposure to
-17.2oC in a controlled environment will reduces the minimum
survival temperature of fully hardened Norstar winter wheat from -24.0 to -17.2oC,
a cold hardiness loss of 6.8oC.

Once vernalization
saturation is complete and the plant enters the reproductive stage, it loses
its ability to cold acclimate (Fowler et al. 1996a) and it will start to
deharden at temperatures warmer than approximately -4oC (Figure 4).
This means that winter wheat will eventually completely deharden once plant
growth resumes in the spring (Figure 1, Fowler et al. 1999). Growth rate and
rate of dehardening are both temperature dependent and because frozen soils
warm slowly in the spring, several weeks of warm air temperatures are required
to re-establish and completely deharden winter cereal plants that have survived
without serious winter damage.